Colon specific gene and protein

ABSTRACT

Human colon specific gene polypeptides and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polynucleotides or polypeptides as a diagnostic marker for colon cancer and as an agent to determine if colon cancer has metastasized. Also disclosed are antibodies specific to the colon specific gene polypeptides which may be used to target cancer cells and be used as part of a colon cancer vaccine. Methods of screening for agonists and antagonists for the polypeptide and therapeutic uses of the antagonists are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Application No. 09/162,508, filed onSep. 29, 1998, which is a divisional of Application No. 08/468,413,filed Jun. 6, 1995, now U.S. Pat. No. 5,861,494, issued on Jan. 19,1999; priority to these applications is hereby claimed under 35 U.S.C. §120, and each of these applications is hereby incorporated by referencein its entirety.

BACKGROUND OF THE INVENTION

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, and the use of such polynucleotides andpolypeptides. The present invention further relates to inhibiting theproduction and function of the polypeptides of the present invention.

The gastrointestinal tract is the most common site of both newlydiagnosed cancers and fatal cancers occurring each year in the USA,figures are somewhat higher for men than for women. The incidence ofcolon cancer in the USA is increasing, while that of gastric cancer isdecreasing, cancer of the small intestine is rare. The incidence ofgastrointestinal cancers varies geographically. Gastric cancer is commonin Japan and uncommon in the United States, whereas colon cancer isuncommon in Japan and common in the USA. An environmental etiologicfactor is strongly suggested by the statistical data showing that peoplewho move to a high-risk area assume the high risk. Some of the suggestedetiologic factors for gastric cancer include aflatoxin, a carcinogenformed by aspergillus flavus and present in contaminated food, smokedfish, alcohol, and Vitamin A and magnesium deficiencies. A diet high infat and low in bulk, and, possibly, degradation products of sterolmetabolism may be the etiologic factors for colon cancer. Certaindisorders may predispose to cancer, for example, pernicious anemia togastric cancer, untreated non-tropical sprue and immune defects tolymphoma and carcinoma, and ulcerative and granulomatous colitis,isolated polyps, and inherited familial polyposis to carcinoma of thecolon.

The most common tumor of the colon is adenomatous polyp. Primarylymphoma is rare in the colon and most common in the small intestine.

Adenomatous polyps are the most common benign gastrointestinal tumors.They occur throughout the GI tract, most commonly in the colon andstomach, and are found more frequently in males than in females. Theymay be single, or more commonly, multiple, and sessile or pedunculated.They may be inherited, as in familial polyposis and Gardener's syndrome,which primarily involves the colon. Development of colon cancer iscommon in familial polyposis. Polyps often cause bleeding, which mayoccult or gross, but rarely cause pain unless complications ensue.Papillary adenoma, a less common form found only in the colon, may alsocause electrolyte loss and mucoid discharge.

A malignant tumor includes a carcinoma of the colon which may beinfiltrating or exophytic and occurs most commonly in the rectosigmoid.Because the content of the ascending colon is liquid, a carcinoma inthis area usually does not cause obstruction, but the patient tends topresent late in the course of the disease with anemia, abdominal pain,or an abdominal mass or a palpable mass.

The prognosis with colonic tumors depends on the degree of bowelwall-invasion and on the presence of regional lymph node involvement anddistant metastases. The prognosis with carcinoma of the rectum anddescending colon is quite unexpectedly good. Cure rates of 80 to 90% arepossible with early resection before nodal invasion develops. For thisreason, great care must be taken to exclude this disease whenunexplained anemia, occult gastrointestinal bleeding, or change in bowelhabits develop in a previously healthy patient. Complete removal of thelesion before it spreads to the lymph nodes provides the best chance ofsurvival for a patient with cancer of the colon. Detection in anasymptomatic patient by occult-bleeding, blood screening results in thehighest five year survival.

Clinically suspected malignant lesions can usually be detectedradiologically. Polyps less than 1 cm can easily be missed, especiallyin the upper sigmoid and in the presence of diverticulosis. Clinicallysuspected and radiologically detected lesions in the esophagus, stomachor colon can be confirmed by fiber optic endoscopy combined withhistologic tissue diagnosis made by directed biopsy and brush sitology.Colonoscopy is another method utilized to detect colon diseases. Benignand malignant polyps not visualized by X-ray are often detected oncolonoscopy. In addition, patients with one lesion on X-ray often haveadditional lesions detected on colonoscopy. Sigmoidoscope examination,however, only detects about 50% of colonic tumors.

The above methods of detecting colon cancer have drawbacks, for example,small colonic tumors may be missed by all of the above-describedmethods, The importance of detecting colon cancer is also extremelyimportant to prevent metastases.

BRIEF SUMMARY OF THE INVENTION

In accordance with an aspect of the present invention, there areprovided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to the RNA transcribed fromthe human colon specific gene of the present invention or to DNAcorresponding to such RNA.

In accordance with another aspect of the present invention there isprovided a method of and products for diagnosing colon cancer metastasesby determining the presence of RNA transcribed from the human colonspecific gene of the present invention or DNA corresponding to such RNAin a sample derived from a host.

In accordance with yet another aspect of the present invention, there isprovided a method of and products for diagnosing colon cancer metastasesby detecting an altered level of a polypeptide corresponding to thecolon specific gene of the present invention in a sample derived from ahost, whereby an elevated level of the polypeptide indicates a coloncancer diagnosis.

In accordance with another aspect of the present invention, there areprovided isolated polynucleotides encoding a polypeptide of the presentinvention, including mRNAs, DNAs, cDNAs, genomic DNAs, as well asantisense analogs and biologically active and diagnostically ortherapeutically useful fragments thereof.

In accordance with a further aspect of the present invention, there areprovided novel polypeptide encoded by the polynucleotides, as well asbiologically active and diagnostically or therapeutically usefulfragments, analogs and derivatives thereof.

In accordance with yet a further aspect of the present invention, thereis provided a process for producing such polypeptides by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a polynucleotide of the presentinvention, under conditions promoting expression of said proteins andsubsequent recovery of said proteins.

In accordance with yet a further aspect of the present invention, thereare provided antibodies specific to such polypeptides.

In accordance with another aspect of the present invention, there areprovided processes for using the polypeptides of the present inventionto treat colon cancer and for using the polypeptide to screen forcompounds which interact with the polypeptides, for example, compoundswhich inhibit or activate the receptor for the polypeptide of thepresent invention.

In accordance with yet another aspect of the present invention, there isprovided a process to screen for compounds which interact with thepolypeptides, for example, compounds which inhibit or activate thepolypeptides of the present invention.

In accordance with yet a further aspect of the present invention, thereare provided processes for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA and manufacture of DNAvectors.

These and other aspects of the present invention should be apparent tothose skilled in the art from the teachings herein.

The following drawings are illustrative of embodiments of the inventionand are not meant to limit the scope of the invention as encompassed bythe claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the cDNA sequence and the corresponding deduced amino acidsequence for the human colon specific gene disclosed in thisapplication. The standard one-letter abbreviations for amino acids areused.

The term “colon specific gene” means that such gene is primarilyexpressed in tissues derived from the colon, and such gene maybe-expressed in cells derived from tissues other than from the colon.However, the expression of such gene is significantly higher in tissuesderived from the colon than from non-colon tissues.

In accordance with an aspect of the present invention, there areprovided isolated nucleic acids (polynucleotides) which encode for themature polypeptide having the deduced amino acid sequence of FIG. 1.(SEQ ID NO:2) or for the mature polypeptide encoded by the cDNA of theclone(s) deposited as ATCC Deposit No. 97129 on Apr. 28, 1995.

The ATCC number referred to above is directed to a biological depositwith the ATCC, 10801 University Boulevard, Manassas, Va. 20110-2209. Thestrain referred to is being maintained under the terms of the BudapestTreaty and will be made available to a patent office signatory to theBudapest Treaty.

A polynucleotide encoding the colon specific gene of the presentinvention was isolated from a human colon cancer cDNA library. Thepolynucleotide contains an open reading frame encoding a protein of 158amino acid residues. The polypeptide exhibits structural homology to agalactose specific lectin from a diamondback rattlesnake with 36%identity and 54% similarity over a 125 amino acid stretch and 30%identity and 52% similarity to a human pancreatic stone proteinprecursor.

The polynucleotides of the present invention may be in the form of RNAor in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptides may beidentical to the coding sequence shown in FIG. 1 (SEQ ID NO:1) or thatof the deposited clone(s) or may be a different coding sequence whichcoding sequence, as a result of the redundancy or degeneracy of thegenetic code, encodes the same mature polypeptides as the DNA of FIG. 1(SEQ ID NO:1) or the deposited cDNA.

The polynucleotides which encode for the mature polypeptide of FIG. 1(SEQ ID NO:2) or for the mature polypeptide encoded by the depositedcDNA may include: only the coding sequence for the mature polypeptide;the coding sequence for the mature polypeptide and additional codingsequence such as a leader or secretory sequence or a proproteinsequence; the coding sequence for the mature polypeptide (and optionallyadditional coding sequence) and non-coding sequence, such as introns ornon-coding sequence 5′ and/or 3′ of the coding sequence for the maturepolypeptides.

Thus, the term “polynucleotide encoding a polypeptide” encompasses apolynucleotide which includes only coding sequence for the polypeptideas well as a polynucleotide which includes additional coding and/ornon-coding sequence.

The present invention further relates to variants of the hereinabovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of thedeposited clone(s). The variants of the polynucleotides may be anaturally occurring allelic variant of the polynucleotides or anon-naturally occurring variant of the polynucleotides.

Thus, the present invention includes polynucleotides encoding the samemature polypeptide as shown in FIG. 1 (SEQ ID NO:2) or the same maturepolypeptide encoded by the cDNA of the deposited clone(s) as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIG. 1 (SEQ ID NO:2) or thepolypeptide encoded by the cDNA of the deposited clone(s). Suchnucleotide variants include deletion variants, substitution variants andaddition or insertion variants.

As hereinabove indicated, the polynucleotides may have a coding sequencewhich is a naturally occurring allelic variant of the coding sequenceshown in FIG. 1 (SEQ ID NO:1) or of the coding sequence of the depositedclone(s). As known in the art, an allelic variant is an alternate formof a polynucleotide sequence which may have a substitution, deletion oraddition of one or more nucleotides, which does not substantially alterthe function of the encoded polypeptide.

The present invention also includes polynucleotides, wherein the codingsequence for the mature polypeptides may be fused in the same readingframe to a polynucleotide sequence which aids in expression andsecretion of a polypeptide from a host cell, for example, a leadersequence which functions as a secretory sequence for controllingtransport of a polypeptide from the cell. The polypeptide having aleader sequence is a preprotein and may have the leader sequence cleavedby the host cell to form the mature form of the polypeptide. Thepolynucleotides may also encode for a proprotein which is the matureprotein plus additional 5′ amino acid residues. A mature protein havinga prosequence is a proprotein and is an inactive form of the protein.Once the prosequence is cleaved an active mature protein remains. Thus,for example, the polynucleotides of the present invention may encode fora mature protein, or for a protein having a prosequence or for a proteinhaving both a prosequence and a presequence (leader sequence).

The polynucleotides of the present invention may also have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as intervening sequences(introns) between individual coding segments (exons).

Fragments of the full length colon specific gene may be used—as ahybridization probe for a cDNA library to isolate the full length geneand to isolate other genes which have a high sequence similarity to thegene or similar biological activity. Probes of this type preferably haveat least 30 bases and may contain, for example, 50 or more bases. Theprobe may also be used to identify a cDNA clone corresponding to a fulllength transcript and a genomic clone or clones that contain thecomplete colon specific gene including regulatory and promotor regions,exons, and introns. An example of a screen comprises isolating thecoding region of the gene by using the known DNA sequence to synthesizean oligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is At least 70%,preferably at least 90%, and more preferably at least 95% identitybetween the sequences. The present invention particularly relates topolynucleotides which hybridize under stringent conditions to thehereinabove-described polynucleotides. As herein used, the term“stringent conditions” means hybridization will occur only if there isat least 95% and preferably at least 97% identity between the sequences.The polynucleotides which hybridize to the hereinabove describedpolynucleotides in a preferred embodiment encode polypeptides whicheither retain substantially the same biological function or activity asthe mature polypeptide encoded by the cDNAs of FIG. 1 (SEQ ID NO:1) orthe deposited cDNA(s).

Alternatively, the polynucleotide may have at least 20 bases, preferably30 bases, and more preferably at least 50 bases which hybridize to apolynucleotide of the present invention and which has an identitythereto, as hereinabove described, and which may or may not retainactivity. For example, such polynucleotides may be employed as probesfor the polynucleotide of FIG. 1 (SEQ ID NO:1), for example, forrecovery of the polynucleotide or as a diagnostic probe or as a PCRprimer.

Thus, the present invention is directed to polynucleotides having atleast a 70% identity, preferably at least 90% and more preferably atleast a 95% identity to a polynucleotide which encodes the polypeptideof FIG. 1 (SEQ ID NO:2) as well as fragments thereof, which fragmentshave at least 30 bases and preferably at least 50 bases and topolypeptides encoded by such polynucleotides.

The deposit(s) referred to herein will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicro-organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence of the polynucleotides contained in the deposited materials, aswell as the amino acid sequence of the polypeptides encoded thereby, areincorporated herein by reference and are controlling in the event of anyconflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

In accordance with another aspect of the present invention there areprovided polynucleotides which are preferably at least 10 base pairs inlength and which hybridize to and are at least 70% identical to RNA (orcorresponding DNA) transcribed from a human gene which has a codingsequence which is at least 90% identical to coding sequence of the DNAsequence of FIG. 1 (SEQ ID NO:1).

Thus, the polynucleotide sequences which hybridize as described abovemay be used to hybridize to and detect the expression of the human geneto which they correspond for use in diagnostic assays as hereinafterdescribed.

In accordance with still another aspect of the present invention thereare provided diagnostic assays for detecting micrometastases of coloncancer in a host. While applicant does not wish to limit the reasoningof the present invention to any specific scientific theory, it isbelieved that the presence of active transcription of a colon specificgene of the present invention in cells of the host, other than thosederived from the colon, is indicative of colon cancer metastases. Thisis true because, while the colon specific gene are found in all cells ofthe body, their transcription to mRNA, cDNA and expression products isprimarily limited to the colon in non-diseased individuals. However, ifcolon cancer is present, colon cancer cells migrate from the cancer toother cells, such that these other cells are now actively transcribingand expressing a colon specific gene at a greater level than is normallyfound in non-diseased individuals, i.e., transcription is higher thanfound in non-colon tissues in healthy individuals. It is the detectionof this enhanced transcription or enhanced protein expression in cells,other than those derived from the colon, which is indicative ofmetastases of colon cancer.

In one example of such a diagnostic assay, an RNA sequence in a samplederived from a tissue other than the colon is detected by hybridizationto a probe. The sample contains a nucleic acid or a mixture of nucleicacids, at least one of which is suspected of containing RNA (orcorresponding cDNA) transcribed from a human colon specific gene of thepresent invention. Thus, for example, in a form of an assay fordetermining the presence of a specific RNA in cells, initially RNA isisolated from the cells.

A sample may be obtained from cells derived from tissue other than fromthe colon including but not limited to blood, urine, saliva, tissuebiopsy and autopsy material. The use of such methods for detectingenhanced transcription to mRNA from a human colon specific gene of thepresent invention or fragment thereof in a sample obtained from cellsderived from other than the colon is well within the scope of thoseskilled in the art from the teachings herein.

The isolation of mRNA comprises isolating total cellular RNA bydisrupting a cell and performing differential centrifugation. Once thetotal RNA is isolated, mRNA is isolated by making use of the adeninenucleotide residues known to those skilled in the art as a poly(A) tailfound on virtually every eukaryotic mRNA molecule at the 3′ end thereof.Oligonucleotides composed of only deoxythymidine [oligo(dT)] are linkedto cellulose and the oligo(dT)-cellulose packed into small columns. Whena preparation of total cellular RNA is passed through such a column, themRNA molecules bind to the oligo(dT) by the poly(A)tails while the restof the RNA flows through the column. The bound mRNAs are then elutedfrom the column and collected.

One example of detecting isolated mRNA transcribed from a colon specificgene of the present invention or a fragment thereof which encodes for apolypeptide of the present invention, comprises screening the collectedmRNAs with specific oligonucleotide probes which have been customdesigned to hybridize to the mRNA to be detected. The oligonucleotideprobe comprises a polynucleotide sequence which hybridizes to at least aportion of the mRNA (or cDNA produced from such RNA) transcribed fromone or more of the colon specific gene of the present invention orfragment thereof. The polynucleotide sequences are at least 70%identical to and hybridize to mRNA (or cDNA produced from such RNA)transcribed from a human colon specific gene of the present inventionhaving exons which includes DNA having at least 90%, preferably at least95% identity and most preferably at least 97% identity to the DNAsequence of FIG. 1 (SEQ ID NO:1).

It is also appreciated that such probes can be and are preferablylabeled with an analytically detectable reagent to facilitateidentification of the probe. Useful reagents include but are not limitedto radioactivity, fluorescent dyes or enzymes capable of catalyzing theformation of a detectable product.

An example of detecting a polynucleotide complementary to the mRNAsequence (cDNA) utilizes the polymerase chain reaction (PCR) inconjunction with reverse transcriptase. PCR is a very powerful methodfor the specific amplification of DNA or RNA stretches (Saiki et al.,Nature, 234:163-166 (1986)). One application of this technology is innucleic acid probe technology to bring up nucleic acid sequences presentin low copy numbers to a detectable level. Numerous diagnostic andscientific applications of this method have been described by H. A.Erlich (ed.) in PCR Technology—Principles and Applications for DNAAmplification, Stockton Press, USA, 1989, and by M. A. Inis (ed.) in PCRProtocols, Academic Press, San Diego, USA, 1990.

RT-PCR is a combination of PCR with the reverse transcriptase enzyme.Reverse transcriptase is an enzyme which produces cDNA molecules fromcorresponding mRNA molecules. This is important since PCR amplifiesnucleic acid molecules, particularly DNA, and this DNA may be producedfrom the mRNA isolated from a sample derived from the host.

A specific example of an RT-PCR diagnostic assay involves removing asample from a tissue of a host. Such a sample will be from a tissue,other than the colon, for example, blood. Therefore, an example of sucha diagnostic assay comprises whole blood gradient isolation of nucleatedcells, total RNA extraction, RT-PCR of total RNA and agarose gelelectrophoresis of PCR products. The PCR products comprise cDNAcomplementary to RNA transcribed from the colon specific gene of thepresent invention or fragments thereof. More particularly, a bloodsample is obtained and the whole blood is combined with an equal volumeof phosphate buffered saline, centrifuged and the lymphocyte andgranulocyte layer is carefully aspirated and rediluted in phosphatebuffered saline and centrifuged again. The supernate is discarded andthe pellet containing nucleated cells is used for RNA extraction usingthe RNazole B method as described by the manufacturer (Tel-Test Inc.,Friendswood, Tex.).

Oligonucleotide primers and probes are prepared with high specificity tothe DNA sequences of the present invention. The probes are at least 10base pairs in length, preferably at least 30 base pairs in length andmay be at least 50 base pairs in length or more. The reversetranscriptase reaction and PCR amplification are performed sequentiallywithout interruption. Taq polymerase is used during PCR and the PCRproducts are concentrated and the entire sample is run on aTris-borate-EDTA agarose gel containing ethidium bromide.

In accordance with another aspect of the present invention, there isprovided a method of diagnosing a disorder of the colon, for examplecolon cancer, by determining altered levels of the colon specificpolypeptides of the present invention in a biological sample, derivedfrom tissue other than from the colon. Elevated levels of the colonspecific polypeptides of the present invention, indicates activetranscription and expression of the corresponding colon specific geneproduct. Assays used to detect levels of a colon specific genepolypeptide in a sample derived from a host are well-known to thoseskilled in the art and include radioimmunoassays, competitive-bindingassays, Western blot analysis, ELISA assays and “sandwich” assays. Abiological sample may include, but is not limited to, tissue extracts,cell samples or biological fluids, however, in accordance with thepresent invention, a biological sample specifically does not includetissue or cells of the colon.

An ELISA assay (Coligan, et al., Current Protocols in Immunology, 1(2),Chapter 6, 1991) initially comprises preparing an antibody specific to acolon specific polypeptide of the present invention, preferably amonoclonal antibody. In addition, a reporter antibody is preparedagainst the monoclonal antibody. To the reporter antibody is attached adetectable reagent such as radioactivity, fluorescence or, in thisexample, a horseradish peroxidase enzyme. A sample is removed from ahost and incubated on a solid support, e.g., a polystyrene dish, thatbinds the proteins in the sample. Any free protein binding sites on thedish are then covered by incubating with a non-specific protein, such asBSA. Next, the monoclonal antibody is incubated in the dish during whichtime the monoclonal antibodies attach to the colon specific polypeptideattached to the polystyrene dish. All unbound monoclonal antibody iswashed out with buffer. The reporter antibody linked to horseradishperoxidase is now placed in the dish resulting in binding of thereporter antibody to any monoclonal antibody bound to the colon specificgene polypeptide. Unattached reporter antibody is then washed out.Peroxidase substrates are then added to the dish and the amount of colordeveloped in a given time period is a measurement of the amount of thecolon specific polypeptide present in a given volume of patient samplewhen compared against a standard curve.

A competition assay may be employed where antibodies specific to a colonspecific polypeptide are attached to a solid support. The colon specificpolypeptide is then labeled and the labeled polypeptide a sample derivedfrom the host are passed over the solid support and the amount of labeldetected, for example, by liquid scintillation chromatography, can becorrelated to a quantity of the colon specific polypeptide in thesample.

A “sandwich” assay is similar to an ELISA assay. In a “sandwich” assay,colon specific polypeptides are passed over a solid support and bind toantibody attached to the solid support. A second antibody is then boundto the colon specific polypeptide. A third antibody which is labeled andis specific to the second antibody, is then passed over the solidsupport and binds to the second antibody and an amount can then bequantified.

In alternative methods, labeled antibodies to a colon specificpolypeptide are used. In a one-step assay, the target molecule, if it ispresent, is immobilized and incubated with a labeled antibody. Thelabeled antibody binds to the immobilized target molecule. After washingto remove the unbound molecules, the sample is assayed for the presenceof the label. In a two-step assay, immobilized target molecule isincubated with an unlabeled antibody. The target molecule-labeledantibody complex, if present, is then bound to a second, labeledantibody that is specific for the unlabeled antibody. The sample iswashed and assayed for the presence of the label.

The choice of marker used to label the antibodies will vary dependingupon the application. However, the choice of marker is readilydeterminable to one skilled in the art. These labeled antibodies may beused in immunoassays as well as in histological applications to detectthe presence of the proteins. The labeled antibodies may be polyclonalor monoclonal.

The presence of active transcription, which is greater than thatnormally found, of the colon specific gene in cells other than from thecolon, by the presence of an altered level of mRNA, cDNA or expressionproducts is an important indication of the presence of a colon cancerwhich has metastasized, since colon cancer cells are migrating from thecolon into the general circulation. Accordingly, this phenomenon mayhave important clinical implications since the method of treating alocalized, as opposed to a metastasized, tumor is entirely different.

The assays described above may also be used to test whether bone marrowpreserved before chemotherapy is contaminated with micrometastases of acolon cancer cell. In the assay, blood cells from the bone marrow areisolated and treated as described above, this method allows one todetermine whether preserved bone marrow is still suitable fortransplantation after chemotherapy.

Antibodies specific to the colon specific polypeptide, for examplemonoclonal antibodies, may also be used to target colon cancer cells,for example, in a method of homing interaction agents which, whencontacting colon cancer cells, destroy them. This is true since theantibodies are specific for colon specific polypeptides which areprimarily expressed in the colon, and a linking of the interaction agentto the antibody would cause the interaction agent to be carried directlyto the prostate.

Antibodies of this type may also be used to do in vivo imaging, forexample, by labeling the antibodies to facilitate scanning of the pelvicarea and the colon. One method for imaging comprises contacting anytumor cells of the colon to be imaged with an anti-colon specificantibody labeled with a detectable marker. The method is performed underconditions such that the labeled antibody binds to any colon specificpolypeptides. In a specific example, the antibodies interact with thecolon, for example, colon cancer cells, and fluoresce upon such contactsuch that imaging and visibility of the colon is enhanced to allow adetermination of the diseased or non-diseased state of the colon.

The present invention further relates to a colon specific genepolypeptide which has the deduced amino acid sequence of FIG. 1 (SEQ IDNO:2) or which have the amino acid sequences encoded by the depositedcDNA(s), as well as fragments, analogs and derivatives of suchpolypeptide.

The terms “fragment,” “derivative” and “analog” when referring to thepolypeptide of FIG. 1 (SEQ ID NO:2) or that encoded by the depositedcDNA(s), means a polypeptide which retains essentially the samebiological function or activity as such polypeptide. Thus, an analogincludes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

The polypeptides of the present invention may be recombinantpolypeptides, natural polypeptides or synthetic polypeptides, preferablyrecombinant polypeptides.

The fragment, derivative or analog of the polypeptides of FIG. 1 (SEQ IDNO:2) or that encoded by the deposited cDNA(s) may be (i) one in whichone or more of the amino acid residues are substituted with a conservedor non-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe polypeptide is fused with another compound, such as a compound toincrease the half-life of the polypeptide (for example, polyethyleneglycol), or (iv) one in which the additional amino acids are fused tothe polypeptide, such as a leader or secretory sequence or a sequencewhich is employed for purification of the mature polypeptide or aproprotein sequence. Such fragments, derivatives and analogs are deemedto be within the scope of those skilled in the art from the teachingsherein.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide ofFIG. 1 (SEQ ID NO:2) (in particular the mature polypeptide) as well aspolypeptides which have at least 70% similarity (preferably at least 70%identity) to the polypeptide of FIG. 1 (SEQ ID NO:2) and more preferablyat least 90% similarity (more preferably at least 90% identity) to thepolypeptide of FIG. 1 (SEQ ID NO:2) and still more preferably at least95% similarity (still more preferably at least 90% identity) to thepolypeptide of FIG. 1 (SEQ ID NO:2) and also include portions of suchpolypeptides with such portion of the polypeptide generally containingat least 30 amino acids and more preferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed ortransfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the colon specific genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothose of ordinarily skill in the art.

The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct mRNAsynthesis. As representative examples of such promoters, there may bementioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgene in prokaryotic or eukaryotic cells or their viruses. The expressionvector also contains a ribosome binding site for translation initiationand a transcription terminator. The vector may also include appropriatesequences for amplifying expression.

In addition, the expression vectors preferably contain one or moreselectable marker gene to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; plant cells, etc. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

More particularly, the present invention also includes recombinantconstructs comprising one or more of the sequences as broadly describedabove. The constructs comprise a vector, such as a plasmid or viralvector, into which a sequence of the invention has been inserted, in aforward or reverse orientation. In a preferred aspect of thisembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P_(R,) P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, or electroporation (Davis, L., Dibner, M., Battey, I.,Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Proteins can be expressed in mammalian cells, yeast, bacteria, or othercells under the control of appropriate promoters. Cell-free translationsystems can also be employed to produce such proteins using RNAs derivedfrom the DNA constructs of the present invention. Appropriate cloningand expression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure ofwhich is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin and adenovirus enhancers.

Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences. Optionally, the heterologous sequence can encodea fusion protein including an N-terminal identification peptideimparting desired characteristics, e.g., stabilization or simplifiedpurification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

As a representative but nonlimiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and bacterial originof replication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). These pBR322 “backbone” sections are combined with an appropriatepromoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of thehost strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Various maumalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts, described by Gluzman,Cell, 23:175 (1981), and other cell lines capable of expressing acompatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

The colon specific gene polypeptide can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

The polynucleotides of the present invention may have the codingsequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. An example ofa marker sequence is a hexa-histidine tag which may be supplied-by avector, preferably a pQE-9 vector, which provides for purification ofthe polypeptide fused-to the marker in the case of a bacterial host, or,for example, the marker sequence may be a hemagglutinin (HA) tag when amammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to-anepitope derived from the influenza hemagglutinin protein (Wilson, I., etal., Cell, 37:76 (1984)).

The polypeptide of the present invention may be a naturally purifiedproduct, or a product of chemical synthetic procedures, or produced byrecombinant techniques from a prokaryotic or eukaryotic host (forexample, by bacterial, yeast, higher plant, insect and mammalian cellsin culture). Depending upon the host employed in a recombinantproduction procedure, the polypeptide of the present invention may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay also include an initial methionine amino acid residue.

In accordance with another aspect of the present invention there areprovided assays which may be used to screen for therapeutics to inhibitthe action of the colon specific gene or colon specific protein of thepresent invention. One assay takes advantage of the reductase functionof these proteins. The present invention discloses methods for selectinga therapeutic which forms a complex with colon specific gene proteinswith sufficient affinity to prevent their biological action. The methodsinclude various assays, including competitive assays where the proteinsare immobilized to a support, and are contacted with a natural substrateand a labeled therapeutic either simultaneously or in either consecutiveorder, and determining whether the therapeutic effectively competes withthe natural substrate in a manner sufficient to prevent binding of theprotein to its substrate.

In another embodiment, the substrate is immobilized to a support, and iscontacted with both a labeled colon specific polypeptide and atherapeutic (or unlabeled proteins and a labeled therapeutic), and it isdetermined whether the amount of the colon specific polypeptide bound tothe substrate is reduced in comparison to the assay without thetherapeutic added. The colon specific polypeptide may be labeled withantibodies.

In another example of such a screening assay, there is provided amammalian cell or membrane preparation expressing a colon specificpolypeptide of the present invention incubated with elements whichundergo simultaneous oxidation and reduction, for example hydrogen andoxygen which together form water, wherein the hydrogen could be labeledby radioactivity, e.g., tritium, in the presence of the compound to bescreened under conditions favoring the oxidation reduction reactionwhere hydrogen and oxygen form water. The ability of the compound toblock this interaction could then be measured.

This invention provides a method for identification of the receptors forthe polypeptide of the present invention. The gene encoding the receptorcan be identified by numerous methods known to those of skill in theart, for example, ligand panning and FACS sorting (Coligan, et al.,Current Protocols in Immun., 1(2), Chapter 5, (1991)). Preferably,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to the polypeptides, and a cDNA library createdfrom this RNA is divided into pools and used to transfect COS cells orother cells that are not responsive to the polypeptides. Transfectedcells which are grown on glass slides are exposed to the labeledpolypeptides. The polypeptides can be labeled by a variety of meansincluding iodination or inclusion of a recognition site for asite-specific protein kinase. Following fixation and incubation, theslides are subjected to autoradiographic analysis. Positive pools areidentified and sub-pools are prepared and retransfected using aniterative sub-pooling and rescreening process, eventually yielding asingle clone(s) that encodes the putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

Further, since the colon specific gene and gene product of the presentinvention is a growth regulator agonist and antagonists to thepolypeptide could be determined by an assay comprising combiningmembrane preparations comprising the receptor for the polypeptide and acompound to be screened and determining the generation of a signal fromthe receptor. In the case of determining an antagonist, the polypeptideof the present invention is added to the assay and the ability of thecompound to compete for receptor sites, i.e., lack of generation ofsignal from receptor, could then be determined.

Potential antagonists to a colon specific polypeptide include antibodiesand anti-idiotypic antibodies as described above, or in some cases, anoligonucleotide, which binds to the polypeptide.

Another potential antagonist is an antisense construct prepared usingantisense technology, which is directed to a colon specificpolynucleotide to prevent transcription. Antisense technology can beused to control gene expression through triple-helix formation orantisense DNA or RNA, both of which methods are based on binding of apolynucleotide to DNA or RNA. For example, the 5′ coding portion of thepolynucleotide sequence, which encodes for the mature polypeptides ofthe present invention, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix -see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervanet al., Science, 251: 1360 (1991)), thereby preventing transcription andthe production of a colon specific polynucleotide. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into the colon specific gene polypeptide(antisense—Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of the colon specific polypeptides.

Potential antagonists also include a small molecule which binds to andoccupies the active site of the colon specific polypeptide therebymaking the active site inaccessible to substrate such that normalbiological activity is prevented. Examples of small molecules includebut are not limited to small peptides or peptide-like molecules.

The antagonists may be employed to treat colon cancer, since theyinteract with the function of colon specific polypeptides in a mannersufficient to inhibit natural function which is necessary for theviability of colon cancer cells. The antagonists may be employed in acomposition with a pharmaceutically acceptable carrier, e.g., ashereinafter described.

The polypeptides and antagonists of the present invention may beemployed in combination with a suitable pharmaceutical carrier. Suchcompositions comprise a therapeutically effective amount of thepolypeptide or antagonist, and a pharmaceutically acceptable carrier orexcipient. Such a carrier includes but is not limited to saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof. The formulation should suit the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepharmaceutical compositions may be employed in conjunction with othertherapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal, intra-anal or intradermalroutes. The pharmaceutical compositions are administered in an amountwhich is effective for treating and/or prophylaxis of the specificindication. In general, they are administered in an amount of at leastabout 10 μg/kg body weight and in most cases they will be administeredin an amount not in excess of about 8 mg/Kg body weight per day. In mostcases, the dosage is from about 10 μg/kg to about 1 mg/kg body weightdaily, taking into account the routes of administration, symptoms, etc.

The colon specific gene polypeptides and antagonists and agonists whichare polypeptides may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as “gene therapy.”

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art. For example, cellsmay be engineered by procedures known in the art by use of a retroviralparticle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding a polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such method should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retrovirus, for example, an adenovirus which may be used toengineer cells in vivo after combination with a suitable deliveryvehicle.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechnicues, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptides.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, ψ-2,ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs.5-14 (1990), which is incorporated herein by reference in its entirety.The vector may transduce the packaging cells through any means known inthe art. Such means include, but are not limited to, electroporation,the use of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

This invention is also related to the use of a colon specific gene ofthe present invention as a diagnostic. For example, some diseases resultfrom inherited defective genes. The colon specific gene of the presentinvention is overexpressed in colon cancer. A mutation in a colonspecific gene of the present invention at the DNA level may be detectedby a variety of techniques. Nucleic acids used for diagnosis (genomicDNA, mRNA, etc.) may be obtained from a patient's cells, other than fromthe colon, such as from blood, urine, saliva, tissue biopsy and autopsymaterial. The genomic DNA may be used directly for detection or may beamplified enzymatically by using PCR (Saiki, et al., Nature, 324:163-166(1986)) prior to analysis. RNA or cDNA may also be used for the samepurpose. As an example, PCR primers complementary to the nucleic acid ofthe instant invention can be used to identify and analyze mutations in acolon specific polynucleotide of the present invention. For example,deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to radiolabelled colonspecific RNA or, alternatively, radiolabelled antisense DNA sequences.

Another well-established method for screening for mutations inparticular segments of DNA after PCR amplification is single-strandconformation polymorphism (SSCP) analysis. PCR products are prepared forSSCP by ten cycles of reamplification to incorporate ³²P-dCTP, digestedwith an appropriate restriction enzyme to generate 200-300 bp fragments,and denatured by heating to 85° C. for 5 min. and then plunged into ice.Electrophoresis is then carried out in a nondenaturing gel (5% glycerol,5% acrylamide) (Glavac, D. and Dean, M., Human Mutation, 2:404-414(1993)).

Sequence differences between the reference gene and “mutants” may berevealed by the direct DNA sequencing method. In addition, cloned DNAsegments may be used as probes to detect specific DNA segments. Thesensitivity of this method is greatly enhanced when combined with PCR.For example, a sequencing primer is used with double-stranded PCRproduct or a single-stranded template molecule generated by a modifiedPCR. The sequence determination is performed by conventional procedureswith radiolabeled nucleotides or by automatic sequencing procedures withfluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments andgels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high-resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g.,Myers, et al., Science, 230:1242 (1985)). In addition, sequencealterations, in particular small deletions, may be detected as changesin the migration pattern of DNA.

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as Rnase and S1 protection or the chemicalcleavage method (e.g., Cotton, et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of the specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing, or the use of restriction enzymes (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blotting.

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with gene associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization. (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 50 or 60 bases. For a review of this technique, see Verma etal., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press,New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between gene anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals bit notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. These antibodies can be, for example, polyclonal ormonoclonal antibodies. The present invention also includes chimeric,single chain, and humanized antibodies, as well as Fab fragments, or theproduct of an Fab expression library. Various procedures known in theart may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, 1975,Nature, 256:495-497), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., 1983, Immunology Today 4:72), and theEBV-hybridoma technique to produce human monoclonal antibodies (Cole, etal., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Transgenic mice mayalso be used to generate antibodies.

The antibodies may also be employed to target colon cancer cells, forexample, in a method of homing interaction agents which, when contactingcolon cancer cells, destroy them. This is true since the antibodies arespecific for the colon specific polypeptides of the present invention. Alinking of the interaction agent to the antibody would cause theinteraction agent to be carried directly to the colon.

Antibodies of this type may also be used to do in vivo imaging, forexample, by labeling the antibodies to facilitate scanning of the pelvicarea and the colon. One method for imaging comprises contacting anycancer cells of the colon to be imaged with an anti-colon specificprotein-antibody labeled with a detectable marker. The method isperformed under conditions such that the labeled antibody binds to thecolon specific polypeptides. In a specific example, the antibodiesinteract with the colon, for example, colon cancer cells, and fluoresceupon contact such that imaging and visibility of the colon are enhancedto allow a determination of the diseased or non-diseased state of thecolon.

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

“Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will legate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated,transformation was performed as described in themethod of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

EXAMPLE 1

Determination of Transcription of the Colon Specific Gene

To assess the presence or absence of active transcription of a colonspecific gene RNA, approximately 6 ml of venous blood is obtained with astandard venipuncture technique using heparinized tubes. Whole blood ismixed with an equal volume of phosphate buffered saline, which is thenlayered over 8 ml of Ficoll (Pharmacia, Uppsala, Sweden) in a 15-mlpolystyrene tube. The gradient is centrifuged at 1800×g for 20 min at 5°C. The lymphocyte and granulocyte layer (approximately 5 ml) iscarefully aspirated and rediluted up to 50 ml with phosphate-bufferedsaline in a 50-ml tube, which is centrifuged again at 1800×g for 20 min.at 5° C. The supernatant is discarded and the pellet containingnucleated cells is used for RNA extraction using the RNazole B method asdescribed by the manufacturer (Tel-Test Inc., Friendswood, Tex.).

To determine the quantity of mRNA from the gene of interest, a probe isdesigned with an identity to the mRNA sequence transcribed from a humangene whose coding portion includes a DNA sequence of FIG. 1. This probeis mixed with the extracted RNA and the mixed DNA and RNA areprecipitated with ethanol −70° C. for 15 minutes). The pellet isresuspended in hybridization buffer and dissolved. The tubes containingthe mixture are incubated in a 72° C. water bath for 10-15 mins. todenature the DNA. The tubes are rapidly transferred to a water bath atthe desired hybridization temperature. Hybridization temperature dependson the G+C content of the DNA. Hybridization is done for 3 hrs. 0.3 mlof nuclease-S1 buffer is added and mixed well. 50 μl of 4.0 M ammoniumacetate and 0.1 M EDTA is added to stop the reaction. The mixture isextracted with phenol/chloroform and 20 μg of carrier tRNA is added andprecipitation is done with an equal volume of isopropanol. Theprecipitate is dissolved in 40 μl of TE (pH 7.4) and run on an alkalineagarose gel. Following electrophoresis, the RNA is microsequenced toconfirm the nucleotide sequence. (See Favaloro, J. et al., MethodsEnzymol., 65:718 (1980) for a more detailed review).

Two oligonucleotide primers are employed to amplify the sequenceisolated by the above methods. The 5′ primer is 20 nucleotides long andthe 3′ primer is a complimentary sequence for the 3′ end of the isolatedmRNA. The primers are custom designed according to the isolated mRNA.The reverse transcriptase reaction and PCR amplification are performedsequentially without interruption in a Perkin Elmer 9600 PCR machine(Emeryville, Calif.). Four hundred ng total RNA in 20 μldiethylpyrocarbonate-treated water are placed in a 65° C. water bath for5 min. and then quickly chilled on ice immediately prior to the additionof PCR reagents. The 50-μl total PCR volume consisted of 2.5 units Taqpolymerase (Perkin-Elmer). 2 units avian myeloblastosis virus reversetranscriptase (Boehringer Mannheim, Indianapolis, Ind.); 200 μM each ofdCTP, dATP, dGTP and dTTP (Perkin Elmer); 18 pM each primer, 10 mMTris-HCl; 50 mM KCl; and 2 mM MgCl₂ (Perkin Elmer). PCR conditions areas follows: cycle 1 is 42° C. for 15 min then 97° C. for 15 s (1 cycle);cycle 2 is 95° C. for 1 min. 60° C. for 1 min, and 72° C. for 30 s (15cycles); cycle 3 is 95° C. for 1 min. 60° C. for 1 min., and 72° C. for1 min. (10 cycles); cycle 4 is 95° C. for 1 min., 60° C. for 1 min., and72° C. for 2 min. (8 cycles); cycle 5 is 72° C. for 15 min. (1 cycle);and the final cycle is a 4° C. hold until sample is taken out of themachine. The 50-μl PCR products are concentrated down to 10 μl withvacuum centrifugation, and a sample is then run on a thin 1.2%Tris-borate-EDTA agarose gel containing ethidium bromide. A band ofexpected size would indicate that this gene is present in the tissueassayed. The amount of RNA in the pellet may be quantified in numerousways, for example, it may be weighed.

Verification of the nucleotide sequence of the PCR products is done bymicrosequencing. The PCR product is purified with a Qiagen PCR ProductPurification Kit (Qiagen, Chatsworth, Calif.) as described by themanufacturer. One μg of the PCR product undergoes PCR sequencing byusing the Taq DyeDeoxy Terminator Cycle sequencing kit in a Perkin-Elmer9600 PCR machine as described by Applied Biosystems (Foster, Calif.).The sequenced product is purified using Centri-Sep columns (PrincetonSeparations, Adelphia, N.J.) as described by the company. This productis then analyzed with an ABI model 373A DNA sequencing system (AppliedBiosystems) integrated with a Macintosh IIci computer.

EXAMPLE 2

Bacterial Expression and Purification of the Colon Specific Gene Proteinand Use For Preparing a Monoclonal Antibody

The DNA sequence encoding a polypeptide of the present invention, ATCC#97129, is initially amplified using PCR oligonucleotide primerscorresponding to the 5′ sequences of the processed protein (minus thesignal peptide sequence) and the vector sequences 3′ to the gene.Additional nucleotides corresponding to the DNA sequence are added tothe 5′ and 3′ sequences respectively. The 5′ oligonucleotide primerGCAGGATCCTGGCTTCCAGAAGCATG (BAMHI) (SEQ ID NO:3) may contain, forexample, a restriction enzyme site followed by nucleotides of codingsequence starting from the presumed terminal amino acid of the processedprotein. The 3′ sequence TACGGGTACCTTGCTCTATGGTCGGTAC (ASP718) (SEQ. IDNO:4) may, for example, contain complementary sequences to a restrictionenzyme site and also be followed by nucleotides of the nucleic acidsequence encoding the protein of interest. The restriction enzyme sitescorrespond to the restriction enzyme sites on a bacterial expressionvector, for example, pQE-32 (Qiagen, Inc. Chatsworth, Calif.). PQE-32encodes antibiotic. resistance (Amp^(r)), a bacterial origin ofreplication (ori), an IPTG-regulatable promoter operator (P/O), aribosome binding site (RBS), a 6-His tag and restriction enzyme sites.pQE-9 is then digested with the restriction enzymes corresponding torestriction enzyme sites contained in he primer sequences. The amplifiedsequences are ligated into pQE-9 and inserted in frame with the sequenceencoding for the histidine tag and the RBS. The ligation mixture is thenused to transform an E. coli strain, for example, M15/rep 4 (Qiagen) bythe procedure described in Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, Cold Spring Laboratory, Press, (1989). M15/rep4contains multiple copies of the plasmid pREP4, which expresses the lacIrepressor and also confers kanamycin resistance (Kan^(r)). Transformantsare identified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis. Clones containing thedesired constructs are grown overnight (O/N) in liquid culture in LBmedia supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/Nculture is used to inoculate a large culture at a ratio of 1:100 to1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) ofbetween 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalacto pyranoside”) isthen added to a final concentration of 1 mM. IPTG induces byinactivating the lacI repressor, clearing the P/O leading to increasedgene expression. Cells are grown an extra 3 to 4 hours. Cells are thenharvested by centrifugation. The cell pellet is solubilized in thechaotropic agent 6 Molar Guanidine HCl. After clarification, solubilizedprotein is purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography411:177-184 (1984)). The protein is eluted from the column in 6 molarguanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3molar guanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione(reduced) and 2 mmolar glutathione (oxidized). After incubation in thissolution for 12 hours the protein is dialyzed to 10 mmolar sodiumphosphate.

The protein purified in this manner may be used as an epitope to raisemonoclonal antibodies specific to such protein. The monoclonalantibodies generated against the polypeptide the isolated protein can beobtained by direct injection of the polypeptides into an animal or byadministering the polypeptides to an animal. The antibodies so obtainedwill then bind to the protein itself. Such antibodies can then be usedto isolate the protein from tissue expressing that polypeptide by theuse of an, for example, ELISA assay.

EXAMPLE 3

Expression Via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over nights After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by the-long terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5′ and 3′ end sequencesrespectively. The 5′ primer contains an EcoRI site and the 3′ primercontains a HindIII site. Equal quantities of the Moloney murine sarcomavirus linear backbone and the EcoRI and HindIII fragment are addedtogether, in the presence of T4 DNA ligase. The resulting mixture ismaintained under conditions appropriate for ligation of the twofragments. The ligation mixture is used to transform bacteria HB101,which are then plated onto agar-containing kanamycin for the purpose ofconfirming that the vector had the gene of interest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

EXAMPLE 4

Cloning and Expression of Colon Specific Gene Polypeptide Using theBaculovirus Expression System

The DNA sequence encoding the full length protein, ATCC #97129, wasamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ sequences of the gene:

The 5′ primer has the sequence 5′ ATCGGGATCCGCCATCATGGCTTCCAGAAGCATGCG(SEQ ID NO:5) and contains a BamHI restriction enzyme site (in bold)followed by 6 nucleotides resembling an efficient signal for theinitiation of translation in eukaryotic cells (Kozak, M., J. Mol. Biol.,196:947-950 (1987) which was just behind the first 20 nucleotides of thecolon specific gene (the initiation codon for translation “ATG” wasunderlined).

The 3′ primer has the sequence 5′ TACGGGTACCTTGCTC TATGGTCGGTAC 3′ (SEQID NO:6) and contains the cleavage site for the restriction endonucleaseAsp718 and 5 nucleotides complementary to the 3′ non-translated sequenceof the colon specific gene. The amplified sequences were isolated from a1% agarose gel using a commercially available kit (“Geneclean,” BIO 101Inc., La Jolla, Calif.). The fragment was then digested with theendonucleases BamHI and Asp718 and then purified again on a 1% agarosegel. This fragment was designated F2.

The vector pA2 (modification of pVL941 vector, discussed below) was usedfor the expression of the colon specific protein using the baculovirusexpression system (for review see: Summers, M. D. and Smith, G. E. 1987,A manual of methods for baculovirus vectors and insect cell cultureprocedures, Texas Agricultural Experimental Station Bulletin NO:1555).This expression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases BamHI andAsp718. The polyadenylation site of the simian virus (SV)40 was used forefficient polyadenylation. For an easy selection of recombinant virusesthe beta-galactosidase gene from E. coli was inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences were flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of cotransfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pRG1 such as pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid was digested with the restriction enzymes and thendephosphorylated using calf intestinal phosphatase by procedures knownin the art. The DNA was then isolated from a 1% agarose gel using thecommercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).This vector DNA was designated V2.

Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNAligase. DH5α cells were then transformed and bacteria identified thatcontained the plasmid (pBac-colon specific polypepitde) with the colonspecific gene using the enzymes BamHI and Asp718. The sequence of thecloned fragment was confirmed by DNA sequencing.

5 μg of the plasmid pBac-colon specific gene was cotransfected with 1.0μg of a commercially available linearized baculovirus (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.) using the lipofectionmethod (Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 μg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac-colonspecific gene were mixed in a sterile well of a microtiter plate-containing 50 μl of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium were added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture was added dropwise to the Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate was rocked back and forthto mix the newly added solution. The plate was then incubated for 27hours at 27° C. After 5 hours the transfection solution was removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum was added. The plate was put back into an incubator andcultivation continued at 27° C. for four days.

After four days the supernatant was collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the viruses were added to the cellsand blue stained plaques were picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses was thenresuspended in an Eppendorf tube containing 200 μl of Grace's medium.The agar was removed by a brief centrifugation and the supernatantcontaining the recombinant baculovirus was used to infect Sf9 cellsseeded in 35 mm dishes. Four days later the supernatants of theseculture dishes were harvested and then stored at 4° C.

Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-colon specific gene at a multiplicity of infection (MOI)of 2. Six hours later the medium was removed and replaced with SF900 IImedium minus methionine and cysteine (Life Technologies Inc.,Gaithersburg). 42 hours later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵Scysteine (Amersham) were added. The colon specific protein was purifiedfrom infected cells 72 hours post infection by cell lysis in hypotonicphosphate buffer and further purified by ion exchange chromatography,size exclusion chromatography, and reverse phase chromatography.

Numerous modifications and variations of the present invention werepossible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1. An isolated monoclonal antibody or portion thereof that specificallybinds to a protein selected from the group consisting of: (a) a proteinwhose sequence consists of amino acid residues 2 to 158 of SEQ ID NO:2;(b) a protein consisting of a fragment of SEQ ID NO:2, wherein saidfragment comprises at least 30 contiguous amino acid residues of SEQ IDNO:2; and (c) a protein consisting of a fragment of SEQ ID NQ:2, whereinsaid fragment comprises at least 50 contiguous amino acid residues ofSEQ ID NO:2.
 2. The antibody or portion thereof of claim 1 thatspecifically binds protein (a).
 3. The antibody or portion thereof ofclaim 1 that specifically binds protein (b).
 4. The antibody or portionthereof of claim 1 that specifically binds protein (c).
 5. The antibodyor portion thereof of claim 1 wherein said protein specifically bound bysaid antibody or portion thereof is glycosylated.
 6. The antibody orportion thereof of claim 1 which is a humanized antibody.
 7. Theantibody or portion thereof of claim 1 which is a human antibody.
 8. Theantibody or portion thereof of claim 1 which is a single chain antibody.9. The antibody or portion thereof of claim 1 which is a Fab fragment.10. The antibody or portion thereof of claim 1 which is labeled.
 11. Anisolated cell that produces the antibody of claim
 1. 12. A hybridomathat produces the antibody of claim
 1. 13. An isolated monoclonalantibody or portion thereof produced by immunizing an animal with aprotein selected from the group consisting of: (a) a protein whosesequence comprises amino acid residues 2 to 158 of SEQ ID NO:2; (b) aprotein whose sequence comprises at least 30 contiguous amino acidresidues of SEQ ID NO:2; and (c) a protein whose sequence comprises atleast 50 contiguous amino acid residues of SEQ ID NO:2, wherein saidantibody or portion thereof specifically binds to the amino acidsequence of SEQ ID NO:2.
 14. The antibody or portion thereof of claim 13produced by immunizing an animal with protein (a).
 15. The antibody orportion thereof of claim 13 by immunizing an animal with protein (b).16. The antibody portion thereof of claim 13 produced by immunizing ananimal with protein (c).
 17. An isolated monoclonal antibody or portionthereof that specifically binds to a protein whose sequence consists ofamino acid residues 1 to 158 of SEQ ID NO:2.
 18. The antibody or portionthereof of claim 17 wherein said protein specifically bound by saidantibody or portion thereof is glycosylated.
 19. The antibody or portionthereof of claim 17 which said humanized antibody.
 20. The antibody orportion thereof of claim 17 which is a humanized antibody.
 21. Theantibody or portion thereof of claim 17 which is a single chainantibody.
 22. The antibody or portion thereof of claim 17 is a Fabfragment.
 23. The antibody or portion thereof of claim 17 which islabeled.
 24. An isolated cell that produces the antibody of claim 17.25. A hybridoma that produces the antibody of claim
 17. 26. An isolatedmonoclonal antibody or portion thereof produced by immunizing an animalwith a protein whose sequence comprises amino acid residues 1 to 158 ofSEQ ID NO:2 wherein said antibody or portion thereof specifically bindsto the protein of SEQ ID NO:2.